Deletion of Double Copies of the US1 Gene Reduces the Infectivity of Recombinant Duck Plague Virus In Vitro and In Vivo

ABSTRACT Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.

replication, viral gene expression, and virus production in vitro and in vivo as well as in pathogenesis in vivo. To this end, the two copies of US1 gene were deleted from DPV and revertant was also generated. Duck embryo fibroblast and ducklings were infected and virus growth was analyzed. The authors concluded based on their results that US1 deletion reduces viral gene expression, replication and virus production both in vitro and in vivo. They also found that in contrast to WT DPV, the US1 deletion mutant virus did not kill ducklings after infection. Their data indicate that US1 is involved in the regulation of virus replication and pathogenesis. The experiments were logically designed, executed, and presented but the study in its current form raises several questions. Comments 1. I strongly recommend that English of the manuscript be thoroughly checked for typos, grammar, words...Also, there are long sentences that need to be broken up. In many cases wrong words are used or words are missing from sentences.
2. The way the study is presented it seems as if they made the double US1 deletion mutant virus in this study despite that it has been published by the same research group 2 years ago (PMID: 32010642). This needs to be addressed. I recommend that they delete and/or re-write the mutant virus generation section addressing what is different here.
3. I am curious whether the deletion of US1 genes affects the neighboring genes US10 and US8, which can influence the interpretation of the phenotype. If US1 deletion also abrogated the expression of US10 and/or US8, they can mask US1 phenotype either reducing or increasing the effect of US1 deletion. 4. It is not clear how the duplicated copies of US1 were deleted. One after the other, that is, that there were 2 subsequent recombinations? The description of bacmid recombination in the Methods section is not understandable.

5.
In the text and figures two groups of ducklings were used for DPV infection, 10 and 6 birds, respectively. In the methods there are 40 and 24 ducklings per group.
6. How the viral DNA copy number was calculated in infected cells? Was it normalized to host DNA? Using different amount of total DNA from different samples will skew the measurement of viral DNA.
7. Figure 4: The difference in viral gene expression between WT and 2deltaUS1 appears to be negligible. I do not consider them substantial changes, which would explain the few fold reduction of virus production. It is unclear what the asterisks mean above the bars. In the figure legend the text says that "differences between two groups were analyzed using Student's t test...". What two groups were compared to each other? The place of asterisk/p-value calculation is incorrect.
8. In the last figure, why different numbers of ducks were used for viral DNA measurement in different tissues according to the graphs? The viral DNA was not tested in the indicated tissues in all infected ducks?
Reviewer #2 (Comments for the Author): The manuscript "Deletion double copies of US1 gene reduce infectivity of recombinant Duck Plague Virus in vitro and in vivo" by Wu et al. assessed the role of the US1 gene in the pathogenesis, replication, and gene expression of duck plague virus (DPV). The authors constructed two recombinant viruses, one with the deletion of the double US1 gene found in DPV, the other a revertant of this deletion. These recombinant viruses were compared in vitro and in vivo for their replication. The double deletion mutant resulted in reduced viral titers in growth kinetics, reduced number and size of plaques, and reduced copies of viral DNA in vitro. This mutant also resulted in reduced lesions and no mortality compared to the revertant and the wildtype control in ducks, with reduced titers in some organs. The manuscript provide interesting insight into the role of the US1 gene in DPV virulence, showing that its deletion results in an attenuated virus that has the potential to be used as a vaccine candidate. However, these findings are masked by the poor quality of the writing and often superficial description of details.

Major comments
The manuscript needs significant editing of the English language. Articles and prepositions are missing throughout the manuscript (e.g., the title should be "Deletion of double copies of US1 gene reduces infectivity of recombinant Duck Plague Virus in vitro and in vivo"), and many sentences are confusing. A few examples are given in the specific comments below, but authors are encouraged to have the text revised by a native speaker or editing service.
The manuscript is not very explicative to a broader audience, with many technical terms not described, and many abbreviations used without explanation (e.g., VZV, PRV, etc) or that need to be better defined, at least the most discussed ones (e.g., ICP22). Although the methods seem to be sound, they are mostly superficially described, and many details are lacking and need to be expanded. More specific comments are below. Additionally, pathological findings are described in a non-technical way, and should be revised by a pathologist to provide correct wording. In addition, some of the statistical analysis seem to be off, with (highly) significant statistical differences found for some graphs that don't seem to have such different values or have large variations (e.g., Figs 3A, 4, 6A).
The discussion is very confusing, and mostly superficial. Not much discussion about the genes that were tested.

Specific comments
The abstract and the Importance are very contrasting -while the abstract emphasizes pathogenesis, the importance is solely focused on vaccine production. Although it is something that could result from this work, production (or testing) of a vaccine was not the point of this manuscript. L13-14: DPV possess a large genome consisting of 78 ORFs. Understanding the function and mechanism of each protein L15-16: US1 is one of the two genes located in the repeat regions of DPV genome, but the function of its encoding protein in DPV L17: Previous studies -please modify throughout L21: that deletion of both copies L22: could represent a potential candidate vaccine L23: remove the word occurrence L30: Change "can overcome the deficiency of them, which will be helpful for the epidemiological" to "can be differentiated from natural infection, which will be helpful for the epidemiological" L32: available against DPV yet L38: alternatively L39: causative agent L54: but is not an IE gene L58: which can directly interact with cyclin-dependent kinase (CDK9) and inhibit the enzyme activity L60: inhibits L64: in involved in virus immune evasion L79: The DPV genome includes a unique long (UL) and unique short (US) region -this description needs to be first mentioned in the previous paragraph L82: US1 gene coexisting. The other duplicated gene is ICP4. L84: we generated a deletion mutant with deletion of the double US1 genes -the way this mutant is termed is often confusing throughout the manuscript. Perhaps the best way to term it is "double US1 gene deletion mutant". I suggest using the abbreviation 2△US1 as often as possible. L90: since results come first, the abbreviations should be spelled out first in the results L94: an example of how the terms used for the mutant are not clear: "double copies of US1 gene deletion strain 2△US1" What is the difference between the wild type and the revertant virus? It would be good to have an explanation of the rationale to have both. L107: was used to indicate L112: These results suggest L124-125: wild type and revertant strains at every time point (Fig.3B). Plaque assays were performed to evaluate L126: US1 gene resulted in smaller L131: "indicating US1 gene promotes the transcription of DPV gene in all phases" -this is confusing. Which genes? Which phases? L134: viral pathogenesis, and the flow chart of the animal experiment is shown in L141: died L145L post-inoculation L146L collected and evaluated for gross and histopathological lesions. L147: without any visible (or noticeable) lesions L160: Livers of birds infected with L161: Histopathological analysis of the spleen showed L171: while DNA quantification of 2△US1 -measuring of genetic material does not necessarily correlate to replication, this was only measured at one time point, so there is no way to know if it increased. L174: The deletion of US1 gene reduced the shedding rate L188-189: An detailed explanation of the mini-F cassette should have been mentioned in the methods section, not only here. L194: resulting in changes L196: restore TK function to study the role L200: viruses are purified L206: by hiding L212: tested L217: but this hypothesis needs further verification L224-225: had significantly reduced gross lesion L226: completely avirulent L236-238: This sentence does not relate much with what was found in the spleen, it should be placed before that sentence about the spleen. L245-246: This sentence is not very coherent. Rephrase it. L249: viral gene transcription in each tissue: This was true even for the spleen, despite no differences in titers. That is something that needs to be discussed. L332: more details about animal experiments are needed -how were animals maintained, N, info about IACUC, species, etc. L337: was MEM administered to the Mock control? L338: When were swabs and tissues collected and what tissues? L341: from cells or tissues were isolated. L357-358: remove the repeated sentence: For extractions of RNA in organs, tissues frozen in liquid nitrogen should be ground into powder with grinding steel balls at first.

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Duck plague virus (DPV) is an alpha-herpesvirus that can cause acute disease (duck plague) with high mortality rates in flocks of ducks, geese and swans. Many of the viral genes in DPV have not been studied regarding what viral genes are essential for replication of the virus in vivo and for pathogenesis. The goal of the study was to test the role of US1 gene of DPV in viral replication, viral gene expression, and virus production in vitro and in vivo as well as in pathogenesis in vivo. To this end, the two copies of US1 gene were deleted from DPV and revertant was also generated. Duck embryo fibroblast and ducklings were infected and virus growth was analyzed. The authors concluded based on their results that US1 deletion reduces viral gene expression, replication and virus production both in vitro and in vivo. They also found that in contrast to WT DPV, the US1 deletion mutant virus did not kill ducklings after infection. Their data indicate that US1 is involved in the regulation of virus replication and pathogenesis. The experiments were logically designed, executed, and presented but the study in its current form raises several questions.
The author ' s response: We thank the reviewer for reading our paper carefully and giving the above positive comments. We have carefully made a comprehensive revision point-by-point in accordance with every comment. Our description on revision according to the comments as follows. Meanwhile, we resubmitted our manuscript with tracked changes that are highlighted in red.
Q1：strongly recommend that English of the manuscript be thoroughly checked for typos, grammar, words...Also, there are long sentences that need to be broken up. In many cases wrong words are used or words are missing from sentences.
The author ' s response: Thanks for the kind comments and suggestions, they are very valuable in improving the quality of our manuscript. We attached great importance to every comment made by you. Therefore, we requested a professional service of English proofreading from American Journal Experts to revise the writing problems of this article. The other grammar and statement errors have been carefully corrected in the revised manuscript, which are highlighted. Meanwhile, we resubmitted our manuscript with tracked changes that are highlighted in red. We hope the revised manuscript can meet the language requirements of Microbiology Spectrum and you can consider giving this manuscript the opportunity to be published in this journal.

Q2：The way the study is presented it seems as if they made the double US1 deletion mutant virus in this study despite that it has been published by the same research group 2 years ago (PMID: 32010642). This needs to be addressed. I recommend that they delete and/or re-write the mutant virus generation section addressing what is different here.
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. As you mentioned, we have constructed a DB-2 △ US1 mutant 2 years ago (PMID: 32010642). However, the major disadvantage of this mutant was the presence of mini-F cassette in TK region. Although the presence of Mini-F cassette in TK gene did not affect the proliferation of the virus on cells in our previously studies, the insertion of Mini-F in TK region on the virus in vivo was not guaranteed, since TK is known to be important for the virulence of herpes virus and no reports of mini-F cassette on virulence have been reported yet. Therefore, to more accurately define the role of US1 gene in the pathogenicity of duck plague virus in vivo, we constructed the 2△US1 and its revertant that did not contain any other mutations except US1 gene on the basis of DB-2△US1(as shown in Figure 1, line 3). In consideration of your comments, we have rewritten the mutant virus generation section and addressed the differences between DB-2 △ US1 and 2 △ US1 in materials, please review the highlighted content in the resubmitted manuscript(line 350-353).

Q3：I am curious whether the deletion of US1 genes affects the neighboring genes US10 and US8, which can influence the interpretation of the phenotype. If US1 deletion also abrogated the expression of US10 and/or US8, they can mask US1 phenotype either reducing or increasing the effect of US1 deletion.
The author ' s response: Thanks very much for your valuable suggestions. According to your suggestion, we tested the integrity of the neighboring genes US10 and US8 by PCR and sequencing. The results were supplemented in resubmitted Figure 2C and corresponding result section. According to the result, we believe that the deletion of US1 gene has no effect on the stability of adjacent genes since no mutations of US10 and US8 gene have been found by PCR and sequencing, which can also indicate that the phenotype variations are only caused by US1 deletion. Please review the highlighted content in the resubmitted manuscript(line 119-123).

Q4：It is not clear how the duplicated copies of US1 were deleted. One after the other, that is, that there were 2 subsequent recombinations? The description of bacmid recombination in the Methods section is not understandable.
The author ' s response: Thank you very much for your insightful comments. The two copies of US1 gene were deleted one by one, and we have obtained 2 subsequent recombinations named BAC-△US1 and BAC-2△US1, the construction details have been described in our previously studies yet(PMID: 32010642). Meanwhile, the focus of this paper is to remove the miniF cassette in DB-△2US1 and restore the US1 gene. Therefore, the deletion of US1 gene has not been described in detail in the method section in this paper. According to your suggestion, we have added a more detailed interpretation regarding the deletion of US1 genes. Please review lines 341-345 and lines 353-360. , 10  and 6 birds, respectively. In the methods there are 40 and 24 ducklings per group. The author's response: Thank you so much for your careful check. We are sorry for the inappropriate statement. We have re-described the grouping of ducks in animal experiments in the method section and made corresponding modifications in Figure 2. Please review lines 425-433.

Q6：How the viral DNA copy number was calculated in infected cells? Was it normalized to host DNA? Using different amount of total DNA from different samples will skew the measurement of viral DNA.
The author's response: Thank you for pointing out this problem of our manuscript. We are sorry for the inappropriate statement. In this paper, the same amount of infected cells or tissues were used for DPV DNA isolation, and the quantification of viral DNA were determined by the TaqMan qRT-PCR which has been established previously in our lab. The accurate copies of DPV DNA was calculated by the standard curve:Y= −4.262X+43.675. Therefore, there was no need to normalize it to host DNA. To be more clearly and in accordance with the reviewer concerns, we have added more detailed interpretations regarding DNA copy number determination method. Please review lines 440-445 of the resubmitted manuscript.

Q7：Figure 4: The difference in viral gene expression between WT and 2deltaUS1 appears to be negligible. I do not consider them substantial changes, which would explain the few fold reduction of virus production. It is unclear what the asterisks mean above the bars. In the figure legend the text says that "differences between two groups were analyzed using Student's t test...". What two groups were compared to each other? The place of asterisk/p-value calculation is incorrect.
The author's response: Thanks for the kind comments and suggestions. Considering our experimental data had at least three groups and other variables were involved, we used One-way and Two-way ANOVA statistical methods to re-analyze our data (Fig3A/D,Fig4,Fig6A/B), and some of the results were different from those previously analyzed by Student's t test. We have re-marked the asterisk in the resubmitted figures based on the re-analyzed result and made corresponding modifications in the figure legends. Please review the resubmitted manuscript and pictures.

Q8：In the last figure, why different numbers of ducks were used for viral DNA measurement in different tissues according to the graphs? The viral DNA was not tested in the indicated tissues in all infected ducks?
The author's response: We are very sorry for our negligence in designing of experiment. Six infected ducklings per group were used for viral DNA and RNA measurement in Fig. 6A and Fig. 6C, and swab samples from 3 ducklings were collected for viral shedding determination. In the design of the experiment, we thought DNA loads in tissues and viral shedding were different indicators of pathogenicity, so we did not consider the use of the same number of duckling samples, which was our negligence. We can re-sample the cloaca for testing if necessary.

The manuscript "Deletion double copies of US1 gene reduce infectivity of recombinant Duck Plague Virus in vitro and in vivo" by Wu et al. assessed the role of the US1 gene in the pathogenesis, replication, and gene expression of duck plague virus (DPV). The authors constructed two recombinant viruses, one with the deletion of the double US1 gene found in DPV, the other a revertant of this deletion. These recombinant viruses were compared in vitro and in vivo for their replication. The double deletion mutant resulted in reduced viral titers in growth kinetics, reduced number and size of plaques, and reduced copies of viral DNA in vitro. This mutant also resulted in reduced lesions and no mortality compared to the revertant and the wildtype control in ducks, with reduced titers in some organs. The manuscript provide interesting insight into the role of the US1 gene in DPV virulence, showing that its deletion results in an attenuated virus that has the potential to be used as a vaccine candidate. However, these findings are masked by the poor quality of the writing and often superficial description of details.
The author ' s response: Thanks very much for your insightful comments and suggestions. They are valuable in improving the quality of our manuscript. According to your suggestions, we have carefully made a comprehensive revision and listed responses point-by-point as follows. Meanwhile, we resubmitted our documents with tracked changes that are highlighted in red.

Major comments： Q1：The manuscript needs significant editing of the English language. Articles and prepositions are missing throughout the manuscript (e.g., the title should be "Deletion of double copies of US1 gene reduces infectivity of recombinant Duck Plague Virus in vitro and in vivo"), and many sentences are confusing. A few examples are given in the specific comments below, but authors are encouraged to have the text revised by a native speaker or editing service.
The author ' s response: Thanks for the kind comments and suggestions, they are very valuable in improving the quality of our manuscript. We attached great importance to every comment made by you. Therefore, we requested a professional service of English proofreading from American Journal Experts to revise the writing problems of this article. The detailed suggestion you mentioned in the title have been revised. The other grammar and statement errors have been carefully corrected in the revised manuscript. Meanwhile, we resubmitted our manuscript with tracked changes that are highlighted in red. We hope the revised manuscript can meet the language requirements of Microbiology Spectrum and you can consider giving this manuscript the opportunity to be published in this journal.

Q2：The manuscript is not very explicative to a broader audience, with many technical terms not described, and many abbreviations used without explanation (e.g., VZV, PRV, etc) or that need to be better defined, at least the most discussed ones (e.g., ICP22).
The author ' s response: We gratefully thanks for the precious time the reviewer spent on our manuscript for making constructive remarks. According to your suggestions, we have carefully redefined all the abbreviations, please review the revised manuscript with tracked changes that are highlighted in red.

Q3： Although the methods seem to be sound, they are mostly superficially described, and many details are lacking and need to be expanded.
The author ' s response: Thanks very much for your valuable suggestions，they are very valuable in improving the quality of our manuscript. Considering your suggestion, we have rewritten some parts of Materials and methods and added more details, please review the revised Materials and methods section in the resubmitted manuscript with tracked changes that are highlighted in red.

Q4：Additionally, pathological findings are described in a non-technical way, and should be revised by a pathologist to provide correct wording.
The author ' s response: Thank you for pointing out this problem in manuscript. We sought help from a pathologist and rewrote the description of the pathological section under his guidance to ensure that it was correct and professional. Please review corresponding paragraph of the resubmitted manuscript with tracked changes that are highlighted in red. (e.g., Figs 3A, 4, 6A).

Q5：In addition, some of the statistical analysis seem to be off, with (highly) significant statistical differences found for some graphs that don't seem to have such different values or have large variations
The author ' s response: Thank you so much for your careful check. In response to your question, We have retested differences between groups again using Student's t test. The test results were found to be the same as before. Considering our experimental data had at least three groups and other variables were involved, we used One-way and Two-way ANOVA statistical methods to re-analyze our data (Fig3A/D,Fig4,Fig6A/B), and some of the results were different from those previously analyzed by Student's t test. We have re-marked the asterisk in the resubmitted figures based on the re-analyzed result and made corresponding modifications in the figure legends. Please review the resubmitted manuscript and pictures.

Q6：The discussion is very confusing, and mostly superficial. Not much discussion about the genes that were tested..
The author's response: Thanks very much for your insightful comments and suggestions. We have extensively revised the discussion section, including why we have to remove min-F cassette from DPV genome, the manipulation of DPV genome on genomic stabililty, and the genes that were tested, etc. Please review the revised manuscript with tracked changes that are highlighted in red(line 219-229, line 244-247 and line 299-311).

Specific comments The abstract and the Importance are very contrasting -while the abstract emphasizes pathogenesis, the importance is solely focused on vaccine production. Although it is something that could result from this work, production (or testing) of a vaccine was not the point of this manuscript.
The author's response: Thank you very much for your insightful comments and valuable suggestions. It really helped us to improve the quality of the manuscript. We agree with your comments and have rewritten the importance section of this manuscript, please review the red highlight content in the resubmitted manuscript.

L15-16: US1 is one of the two genes located in the repeat regions of DPV genome, but the function of its encoding protein in DPV L17: Previous studies -please modify throughout L21: that deletion of both copies
L22: could represent a potential candidate vaccine L23: remove the word occurrence L30: Change "can overcome the deficiency of them, which will be helpful for the epidemiological" to "can be differentiated from natural infection, which will be helpful for the epidemiological" L32: available against DPV yet L38: alternatively L39: causative agent (Fig.3B) The author ' s response: Thank you very much for your insightful comments and valuable suggestions. We have carefully made a comprehensive revision based on your comment and suggestion, and the above writing problems you mentioned have been modified and checked by a professional service of English proofreading from American Journal Experts. Please review the red highlight content in the resubmitted manuscript.

L249: viral gene transcription in each tissue: This was true even for the spleen, despite no differences in titers. That is something that needs to be discussed.
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. Considering your suggestion, we have discussed that viral gene transcription reduced in spleen, please review the corresponding section with red highlighted track in the resubmitted manuscript(line 290-298 ).

L332: more details about animal experiments are needed -how were animals maintained, N, info about IACUC, species, etc.
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. We have added more details about animal species, numbers, maintained, ect. in the materials section. Please review the red highlight content in the resubmitted manuscript(line 420-425 ).

L337: was MEM administered to the Mock control?
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. Yes, MEM was used as a mock control for animal experiment, we have added a footnote of it. please review the resubmitted manuscript.

L338: When were swabs and tissues collected and what tissues?
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. As shown in Fig5A, 24 ducklings (six per group) were sacrificed at 5 days post infection, swabs and tissues(live, spleen and duodenum ) were collected at this time point for the lesions, viral loads and gene expression analysis. We have added the details in materials, please review the resubmitted manuscript in highlighted content(line 429-433).

L341: from cells or tissues were isolated. L357-358: remove the repeated sentence: For extractions of RNA in organs, tissues frozen in liquid nitrogen should be ground into powder with grinding steel balls at first
The author's response: Thank you very much for your insightful comments and valuable suggestions. We have carefully made a comprehensive revision based on your comment and suggestion, and the above writing problems you mentioned have been modified and checked by a professional service of English proofreading from American Journal Experts. Please review the red highlight content in the resubmitted manuscript.

The manuscript has been significantly improved. The authors addressed all of my concerns except one. It was asked if US1 deletion affects the expression of neighboring genes US8 and/or US10. This was not answered. They sequenced US8 and US10 and found no alteration, which was expected. But I was asking about their gene (mRNA) expression.
The author ' s response: We thank the reviewer for reading our manuscript carefully and giving the above positive comments for our revised article. According to your suggestion, we detected the expression of neighboring genes US10 and US8 by RT-qPCR at different time points after infection. As expected, the expression of US10 and US8 genes in the 2△ US1 strain decreased significantly at the early stage of infection compared with that of the wild strain, but gradually recovered as the infection time progressed. Combined with the results of sequencing, we believed that the decreased expression levels of US10 and US8 genes were caused by the transcriptional regulation of US1 gene, and had nothing to do with its genome stability. The results were attached in the supplementary figures( Figure S1). Meanwhile, we resubmitted our manuscript with tracked changes that are highlighted in red.

Review 2#:
The revised article is significantly improved, much clearer and better-written. The English language improved considerably. The authors have addressed my overall concerns. However, due to the extensive number of changes done to the manuscript, there were some minor concerns found as listed below.
The author ' s response: Thanks very much for your positive comments for our revised article and your valuable suggestions. They are valuable in improving the quality of our manuscript. According to your suggestions, we have carefully made a comprehensive revision and listed responses point-by-point as follows. Meanwhile, we resubmitted our documents with tracked changes that are highlighted in red.

minor concerns：
L86-87: suggesting that it could be a promising candidate vaccine strain that can help control duck plague outbreaks. L92-96: although the description is much improved, this sentence is too long and should be broken up in two or more sentences. L173: showed obvious pathological findings. The term symptoms is not correct for animals, and here it is not description of clinical signs, but of pathological findings. L179-180: After 5 days post inoculation, the 2△US1 showed mild inflammatory... L187: The duodenum showed mild necrosis in the mucosal epithelium L198: Surprisingly, we found no significant difference in viral loads L200-202: This sentence is confusing and a bit overinterpreting. What can be said by results is that 2△US1 showed reduced copy numbers in cloacal samples, suggesting lower shedding of virus. By looking at copy numbers alone it cannot be inferred that infection (colonization) was reduced and clearance increased. L213-216: This sentence about the constructions is very lengthy and should be broken up. This is true for many other sentences in the discussion (e.g., L230-233, L244-248, L248-252, L255-259, L253-255: This sentence is confusing and not well-written. L280: 2△US1 showed attenuated virulence L335: earlier by our group The author ' s response: Thank you very much for your insightful comments and valuable suggestions. We have carefully made a comprehensive revision based on your comment and suggestion, and the above writing problems you mentioned have been modified and checked. Please review the red highlight content in the resubmitted manuscript.
L282: "but were not completely avirulent to the tested ducklings" -this sentence is not clear, do the authors mean "although 2△US1 was not completely avirulent in inoculated duclings"?
The author ' s response: Thank you very much for your insightful comments and valuable suggestions. We are sorry for the inappropriate statement. To make it clear, we have changed the sentence to "but it was still mildly pathogenic to the ducklings". Please review line 283 of the resubmitted manuscript. L285: what does "ocular lesions of the liver and thymus" mean? Is this referring to macroscopic or gross lesions?
The author's response: Thank you very much for your insightful comments and valuable suggestions. Yes, the sentence "ocular lesions of the liver and thymus" mean the macroscopic lesions. We have changed the sentence to "macroscopic lesions" to make it clear. Please review line 286 of the resubmitted manuscript. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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